http://www.omicsonline.org/paahome.

php
Ketola Raimo Allan,
University of Helsinki,
Finland
Joseph P. Fuhr,
Widener University, UK
Zhe-Sheng (Jason)
Chen, St. Johns
University, USA
Alexander Panossian,
Swedish Herbal Institute,
Sweden
Bruno Sarmento,
University of Porto,
Portugal
Ana Lucia Abujamra,
Federal University of Rio
Grande do Sul, Brazil
Praveen S. Hiremath,
Pharmaceutics
International Inc., USA
Jun Zhang,
Development Sciences
of Abbott Laboratories,
USA
Viswatej Vemulapalli,
Pharmaceutics
International Inc., USA
Subhash C. Chauhan,
University of South
Dakota, USA
Josef Ozer, Pzer
Biotech, USA
Lee Jia, National
Cancer Institute/
NIH, USA
P
harmaceutical Science is a fast
growing area of drug research
which includes Drug discovery, Drug
development and mapping their effects
in animals and people to nd remedy to
a particular disease.
Being an Open Access journal, it makes
the research freely available to the
public for greater global exchange of
knowledge in the chemical sciences and
pharmacological areas.
It encourages research in Drug
discovery and Development by creating
awareness about the chemistry of
substances, interaction with the body,
metabolized and eliminated inorder to
develop drugs in an unique way.
It aims to provide latest practical
technology to the readers of various
categories i.e., scientists, clinicians,
researchers, organizations and general
public to sp read the latest research on
drug discovery.
Pharmaceutica Analytica Acta - Open Access
using online manuscript submission, review
and tracking systems of Editorial Manager for
quality and quick review processing.
Submit your manuscript at
http://www.omicsonline.org/submission
ISSN: 2153-2435



































O
M
I
C
S
P
u
blishin
g
G
r
o
u
p
OMICS Publishing Group
5716 Corsa Ave., Suite 110, Westlake, Los Angeles, CA 91362-7354, USA, Phone: +1- 650-268-9744, Fax: +1-650-618-1414, Toll free: +1-800-216-6499
OMICS Publishing Group
Pharm Anal Acta
ISSN: 2153-2435 PAA, an open access journal
Pharmaceutica Analytica Acta - Open Access
www.omicsonline.org
Research Article
OPEN ACCESS Freely available online
doi:10.4172/2153-2435.1000113
Volume 1 Issue 21000113
Simultaneous HPLC Analysis of Betamethasone and
Clotrimazole in Cream Formulations
Adnan Manassra
1
*, Mustafa Khamis
1
, Magdy el-Dakiky
1
, Zuhair Abdel-Qader
2
and Fuad Al-Rimawi
1
1
Faculty of Science and Technology, Al-Quds University, P.O. Box 20002, East Jerusalem
2
Research and Development Department, Jerusalem Pharmaceutical Co., P.O. Box 3570, Al-Bireh, Palestine
Keywords: HPLC; Betamethasone; Clotrimazole; Pharmaceutical
preparations
Introduction
Betamethasone is a potent synthetic glucocorticoid that is widely
used for the treatment of inflammation, allergies and other diseases
related to glucocorticoid deficiency [1]. Clotrimazole is a chlorinated
synthetic imidazole derivative having antifungal and antibacterial
activities, which are used in the treatment of some infections [2].
The combination of betamethasone and clotrimazole is used for the
treatment of candidiasis, vulvovaginal candidiasis and other species
of Candida [3-5] and provides anti-inflammatory action.
In the scientific literature, analysis of betamethasone and
clotrimazole has been reported as individual ingredients [1-2,6-
14] and in combination products [15]. Betamethasone has been
determined in different pharmaceutical preparations by HPLC [1,6-
7]. Clotrimazole has been determined in different pharmaceutical
preparations by: Titration method [2], gas liquid chromatography
[8], high performance TLC (HPTLC) [9], micellar electrokinetic
chromatography (MEKC) [10] and by HPLC [11-14]. Reversed-phase
LC for the simultaneous determination of betamethasone and
clotrimazole in cream formulations has been described in the USP
[15]. However, sample preparation of the cream in this USP method
is time consuming (about one hour), tedious (requires centrifuge and
heating). The main objective of this study is, therefore, to develop
and validate an HPLC method involving minimum sample preparation,
good resolution, reasonable analysis time, good accuracy, high
precision, good specificity, good linearity, and excellent reliability.
Material and Methods
Equipments and settings
The HPLC measurements were carried out using a Merck Hitachi
HPLC (Hitachi, Ltd. Tokyo, Japan) equipped with a manual loop
injector that was connected to a photo diode array detector, and a
recorder.
An analytical column with C18 stationary phase (250 X 4.0mm
i.d.) bonded onto 5m silica gel manufactured by Merck (Darmstadt,
Germany) was used for chromatographic separation. Degassing of
the mobile phase was performed using Sonnicator (Fisher Scientific
FS 220). Instrumental HPLC settings were as follows: flow rate 1.5
ml/min; injection volume 5l, column temperature ambient; and
wavelength 254nm.
*Corresponding author: Adnan Manassra, Faculty of Science and Technology,
Al-Quds University, P.O. Box 20002, East Jerusalem, E-mail: amanassra@yahoo.
com
Received August 30, 2010; Accepted October 11, 2010 Published December
30, 2010
Citation: Manassra A, Khamis M, el-Dakiky M, Abdel-Qader Z, Al-Rimawi F (2010)
Simultaneous HPLC Analysis of Betamethasone and Clotrimazole in Cream
Formulations. Pharm Anal Acta 1:113. doi:10.4172/2153-2435.1000113
Copyright: 2010 Manassra A, et al. This is an open-access article distributed
under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the
original author and source are credited.
Abstract
An HPLC method for the simultaneous quantitative determination of betamethasone and clotrimazole in cream
formulation has been developed. The method utilizes a reversed-phase C18 (250 X 4.0 mm) stationary phase, with a
mixture of methanol-acetate buffer-acetonitrile (33:27:40, v/v) as a mobile phase, and spectrophotometric UV detection
at 254 nm. The method has been validated for cream formulations containing betamethasone and clotrimazole with
linear range of 0.025 to 0.075 mg/ml for betamethasone with a correlation coeffcient of 0.9996, and linear range of
0.25 to 0.75 mg/ml for clotrimazole with a correlation coeffcient of 1.000. The results demonstrated that this method is
accurate, precise, specifc, linear, reliable, sensitive, and fast.
Reagents
All the active and inactive ingredients of the cream were kindly
supplied by Jerusalem Pharmaceuticals Co. Ltd., Al-Bireh, Palestine, and
were of British Pharmacopoeia (BP) or Untied States Pharmacopoeia
(USP) quality, and were used without further purification. Acetonitrile
and methanol (HPLC grade) are from J. T Baker (NJ, USA). All other
chemicals were of analytical reagent grade and they are from Merck
(Darmstadt, Germany). Water used was distilled and deionised by
passing through water purification system.
Acetate buffer with a pH of 6.8 was prepared by dissolving 25.0
g ammonium acetate in 1000 ml of distilled deionised water. Diluent
was prepared by mixing 990 ml of methanol and 10 ml of acetic acid.
The mobile phase, standard and sample solutions were filtered using
0.45m microporous filters type polyamid.
Standards and sample preparation
A standard solution having 0.05 mg/ml and 0.5 mg/ml of
betamethasone and clotrimazole, respectively was prepared as
follows: 100 mg of betamethasone was dissolved in 100 ml diluent
(Solution A), 50 mg of clotrimazole was dissolved in 10 ml diluent
(Solution B). Then, 5 ml of Solution A and 10 ml of Solution B was
diluted to 100 ml with diluent.
The sample was prepared by weighing 5.0 g of the cream which is
equivalent to 5.0 mg of betamethasone and 50.0 mg of clotrimazole
in a100 ml-beaker, and then an adequate volume of diluent was
added with stirring until homogeneous solution was obtained. The
solution was transferred to a 100 ml volumetric flask and the volume
was completed to 100 ml with diluent.
Citation: Manassra A, Khamis M, el-Dakiky M, Abdel-Qader Z, Al-Rimawi F (2010) Simultaneous HPLC Analysis of Betamethasone and Clotrimazole
in Cream Formulations. Pharm Anal Acta 1:113. doi:10.4172/2153-2435.1000113
OMICS Publishing Group
Pharm Anal Acta
ISSN: 2153-2435 PAA, an open access journal
Page 2 of 3
Volume 1 Issue 21000113
Solutions for validation study
Linearity and range: Stock standard solution having 0.5 mg/ml
and 5.0 mg/ml of betamethasone and clotrimazole, respectively
was prepared by dissolving 50 mg of betamethasone and 500 mg
of clotrimazole in 100 ml diluent. Five different concentrations of
betamethasone and clotrimazole were prepared from the sock
solution as follows: 5 ml of stock solution was diluted to 100 ml
with diluent (0.025 mg/ml of betamethasone and 0.25 mg/ml of
clotrimazole), 15 ml of stock solution was diluted to 200 ml with
diluent (0.0375 mg/ml of betamethasone and 0.375 mg/ml of
clotrimazole), 10 ml of stock solution was diluted to 100 ml with
diluent (0.05 mg/ml of betamethasone and 0. 5 mg/ml of clotrimazole),
25 ml of stock solution was diluted to 200 ml with diluent (0.0625
mg/ml of betamethasone and 0.625 mg/ml of clotrimazole), and 15 ml
of stock solution was diluted to 100 ml with diluent (0.075 mg/ml of
betamethasone and 0.75 mg/ml of clotrimazole).
Accuracy (Recovery): For recovery study, the placebo of the
cream formulation was prepared according to the formulation
procedure. Then, to the required quantity of the placebo, a known
quantity of betamethasone and clotrimazole was added to get three
concentration levels of betamethasone and clotrimazole (50%, 100%,
and 150% of the working concentration of betamethasone and
clotrimazole).
Results and Discussion
Method development
Reversed-phase LC-method was employed for the
chromatographic separation of betamethasone and clotrimazole.
To this end, reversed-phase C8 and C18 columns using mixture of
organic solvents (acetonitrile, and methanol) and aqueous buffer as
a mobile phase was tested. While C8 column does not show enough
resolution between these two analytes, C18 column shows adequate
resolution. In order to optimize the chromatographic parameters, the
effects of the buffer, methanol, and acetonitrile volume fractions on
the separation of betamethasone and clotrimazole were studied. The
optimum composition of the mobile phase was selected based on
obtaining stable baseline, sharp peaks in reasonable time, and good
separation of betamethasone and clotrimazole from each other and
from the excipients present in the cream formulation. The selected
composition was methanol/acetate buffer (pH = 6.8)/acetonitrile
with a ratio of 33:27:40 by volume. A typical chromatogram
of betamethasone and clotrimazole (prepared from the cream
preparation) is shown in Figure 1.
Method validation
After method development, validation of the current method
was performed in accordance with USP requirements for assay
determination (Category-I: Analytical methods for quantitation of
active ingredients in finished pharmaceutical products) which include
accuracy, precision, selectivity, linearity and range.
Linearity and range: To evaluate linearity of the method, five
calibration standards of betamethasone and clotrimazole containing
0.025 to 0.075 mg/ml of betamethasone and 0.25 to 0.75 mg/ml of
clotrimazole were analyzed. A plot of peak area vs. amount injected
was linear in the range of 0.025 to 0.075 mg/ml of betamethasone
with a correlation coefficient of 0.9996, and in the range of 0.25 to
0.75 mg/ml of clotrimazole with a correlation coefficient of 1.000.
Accuracy (Recovery): Percentage recovery of betamethasone and
clotrimazole using this method was determined by analyzing the
three samples of the cream (prepared as in section 2.4.2) and the
percentage of betamethasone and clotrimazole in the samples was
calculated at the three concentration levels (50%, 100%, and 15%), by
simple proportion from peak areas of the sample and a standard.
Results have shown that the mean recovery of betamethasone and
clotrimazole is within 100 2.0%, see Table 1.
Precision: Instrumental precision of this method was determined
by injecting the standard solution of the two analytes six times. The
RSD of peak areas of betamethasone and clotrimazole for the six
replicates was found to be 0.73% and 0.52% for betamethasone and
clotrimazole, respectively.
Intermediate-precision of the method was also evaluated by
analyzing six samples of the two analytes at six days. Results which
are represented in Table 1 show good intermediate-precision of the
method (average percentage is 98.7% and 99.0% for betamethasone,
and clotrimazole, respectively). Furthermore, RSD of the four samples
was found to be less than 1.0%, see Table 2.
Selectivity: Selectivity of the current method was demonstrated
by good separation of betamethasone and clotrimazole. Furthermore,
betamethasone and clotrimazole are good separated from the
excipients of the cream preparation as seen in Figure 1.
% recovery
Concentration level Betamethasone Clotrimazole
50 99.6 101.0
100 100.3 100.6
150 98.6 99.8
Average: 99.5 Average: 100.5
Table 1: percentage recovery of betamethasone and clotrimazole at three
concentration levels.
Table 2: Intermediate-precision of the method for analyzing betamethasone and
clotrimazole in cream formulations.
*Mean R.S.D. for four samples
Day % Betamethasone* % Clotrimazole*
1 98.30.86 99.70.52
2 98.60.59 98.30.65
3 98.20.50 99.30.77
4 99.20.62 98.60.34
5 98.50.78 98.50.70
6 99.30.89 99.30.72
B
C
b
a
I
n
t
e
n
s
i
t
y

(
A
U
)
Retention Time (min)
0.12
0.10
0.08
0.06
0.04
0.02
0.00
0.0 1 2 3 4 5 6 7 8 9 10
1
.
1
1
1
.
7
2
2
.
6
0
2
.
0
4
6
.
9
5
Figure 1: Typical chromatogram of pharmaceutical combination containinig
0.05 mg/ml of betamethasone, and 0.5 mg/ml of clotrimazole. Column: C18
(25cm X 4.0mm i.d). Mobile phase: methanol/buffer/acetonitrile (33:27:40, v/v).
Flow rate: 1.5ml/min; : 254 nm. Peaks identifcation, B: Betamethasone, C:
Clotrimazole, a, b: Excipients from the cream preparation.
Citation: Manassra A, Khamis M, el-Dakiky M, Abdel-Qader Z, Al-Rimawi F (2010) Simultaneous HPLC Analysis of Betamethasone and Clotrimazole
in Cream Formulations. Pharm Anal Acta 1:113. doi:10.4172/2153-2435.1000113
OMICS Publishing Group
Pharm Anal Acta
ISSN: 2153-2435 PAA, an open access journal
Page 3 of 3
Volume 1 Issue 21000113
Conclusion
The method represents a fast analytical procedure for
simultaneous determination of betamethasone and clotrimazole in
cream formulations with good accuracy, precision, reproducibility,
linearity, selectivity, and reliability. The sample preparation is simple,
and the elution is isocratic. The method is amenable to the analysis
of large number of samples with excellent precision and accuracy.
Acknowledgments
We would like to thanks Jerusalem Pharmaceuticals Company for their
encouragement, cooperation, help and providing all facilities.
References
1. Liu K, Chen S, Wu S, Kou H, Wu H (2004) HPLC determination of Betamethsone
and Dexamethasone. J Chromatogr A 676: 455-460.
2. Massaccesi M (1986) Two-phase Titration of some Imidazole derivatives in
pharmaceutical preparations; Analyst 111: 987-989.
3. British Pharmacopoeia Commission Offce, British Pharmacopoeia 1998, the
stationary offce limited, London P 369.
4. Sawyer PR, Brogden RN, Pinder RM, Speight TM, Avery (1975) Clotrimazole:
a review of its antifungal activity and therapeutic effcacy. Drugs 9: 424-447.
5. Lehne RA, Crosby LJ, Hamilton DB, Moore LA (1990) Pharmacology WB
Saunders Company 746.
6. The United States Pharmacopoeia (1995) The National Formulary USP-23-
NF18 Pharmacopeial Convention, Inc Rockville 187.
7. Wang L, Yang YY, Chung TS, Chen XQ (2002) Determination of betamethasone
disodium phosphate in the in-vitro media of PLGA microspheres by high-
performance liquid chromatography. J Pharm Biomed Anal 28: 629-635.
8. Wallace SM, Shah VP, Riegelman S, Epstein WL (1978) Electron capture Gas
Chromatographic assay for Miconazole and Clotrimazole in skin samples. Anal
Lett B 11: 461-468.
9. Vaidya VV, Menon SN, Singh GR, Kekare MB, Choukekar MP (2007)
Simultaneous HPTLC determination of clotrimazole and tinidazole in a
pharmaceutical formulation. J Planar Chromator Modern TLC 20: 145-147.
10. Hamoudova R, Pospisilova M, Kavalirova A, Solich P, Sicha J (2006) Separation
and determination of clotrimazole, methylparaben and propylparaben in
pharmaceutical preparation by micellar electrokinetic chromatography. J Pharm
Biomed Anal 40: 215-219.
11. Cavrini V, Di Pietra AM, Raggi MA (1982) HPLC analysis of imidazole
antifungals in commercial dosage forms. Int J Pharm 10: 119-124.
12. Hoogerheide JG, Strusiak SH, Taddei CR, Townley ER, Wyka BE (1981) HPLC
determination of Clotrimazole in pharmaceutical formulations. J Assoc Off Anal
Chem 64: 864-869.
13. Guifang W, Xia S, Shouyao Z, Lihong Z (1996) Determination of Clotrimazole
and Dyclonine HCl in compound Clotrimazole cream by HPLC. Zhongguo
Yiyuan Yaoxue Zazhi 16: 66-67.
14. Tendolkar NM, Desai BS, Shinde VM (1994) Simultaneous determination of
Tinidazole and Clotrimazole from tablets by RP-HPLC. Indian Drugs 31: 551-
553.
15. The United States Pharmacopoeia (2009) The National Formulary.
Pharmacopeial Convention Inc Rockville.